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The anaphase-promoting complex/cyclosome activator Cdh1 modulates Rho GTPase by targeting p190 RhoGAP for degradation.

ABSTRACT: Cdh1 is an activator of the anaphase-promoting complex/cyclosome and contributes to mitotic exit and G(1) maintenance by targeting cell cycle proteins for degradation. However, Cdh1 is expressed and active in postmitotic or quiescent cells, suggesting that it has functions other than cell cycle control. Here, we found that homozygous Cdh1 gene-trapped (Cdh1(GT/GT)) mouse embryonic fibroblasts (MEFs) and Cdh1-depleted HeLa cells reduced stress fiber formation significantly. The GTP-bound active Rho protein was apparently decreased in the Cdh1-depleted cells. The p190 protein, a major GTPase-activating protein for Rho, accumulated both in Cdh1(GT/GT) MEFs and in Cdh1-knockdown HeLa cells. Cdh1 formed a physical complex with p190 and stimulated the efficient ubiquitination of p190, both in in vitro and in vivo. The motility of Cdh1-depleted HeLa cells was impaired; however, codepletion of p190 rescued the migration activity of these cells. Moreover, Cdh1(GT/GT) embryos exhibited phenotypes similar to those observed for Rho-associated kinase I and II knockout mice: eyelid closure delay and disruptive architecture with frequent thrombus formation in the placental labyrinth layer, respectively. Furthermore, the p190 protein accumulated in the Cdh1(GT/GT) embryonic tissues. Our data revealed a novel function for Cdh1 as a regulator of Rho and provided insights into the role of Cdh1 in cell cytoskeleton organization and cell motility.

SUBMITTER: Naoe H

PROVIDER: S-EPMC2916440 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Acm1 is a negative regulator of the CDH1-dependent anaphase-promoting complex/cyclosome in budding yeast.

Project description:Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/CCdh1 modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G1 and disappearing in late M. In acm1Delta strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/CCdh1-mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1Delta cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1Delta and hsl1Delta resulted in a dramatic enhancement of bud elongation and G2/M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/CCdh1.

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The PAR-6 polarity protein regulates dendritic spine morphogenesis through p190 RhoGAP and the Rho GTPase.

Project description:The majority of excitatory synaptic transmission in the brain occurs at dendritic spines, which are actin-rich protrusions on the dendrites. The asymmetric nature of these structures suggests that proteins regulating cell polarity might be involved in their formation. Indeed, the polarity protein PAR-3 is required for normal spine morphogenesis. However, this function is independent of association with atypical protein kinase C (aPKC) and PAR-6. Here we show that PAR-6 together with aPKC plays a distinct but essential role in spine morphogenesis. Knockdown of PAR-6 inhibits spine morphogenesis, whereas overexpression of PAR-6 increases spine density, and these effects are mediated by aPKC. Using a FRET biosensor, we further show that p190 RhoGAP and RhoA act downstream of the PAR-6/aPKC complex. These results define a role for PAR-6 and aPKC in dendritic spine biogenesis and maintenance, and reveal an unexpected link between the PAR-6/aPKC complex and RhoA activity.

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Developmental role and regulation of cortex, a meiosis-specific anaphase-promoting complex/cyclosome activator.

Project description:During oogenesis in metazoans, the meiotic divisions must be coordinated with development of the oocyte to ensure successful fertilization and subsequent embryogenesis. The ways in which the mitotic machinery is specialized for meiosis are not fully understood. cortex, which encodes a putative female meiosis-specific anaphase-promoting complex/cyclosome (APC/C) activator, is required for proper meiosis in Drosophila. We demonstrate that CORT physically associates with core subunits of the APC/C in ovaries. APC/C(CORT) targets Cyclin A for degradation prior to the metaphase I arrest, while Cyclins B and B3 are not targeted until after egg activation. We investigate the regulation of CORT and find that CORT protein is specifically expressed during the meiotic divisions in the oocyte. Polyadenylation of cort mRNA is correlated with appearance of CORT protein at oocyte maturation, while deadenylation of cort mRNA occurs in the early embryo. CORT protein is targeted for degradation by the APC/C following egg activation, and this degradation is dependent on an intact D-box in the C terminus of CORT. Our studies reveal the mechanism for developmental regulation of an APC/C activator and suggest it is one strategy for control of the female meiotic cell cycle in a multicellular organism.

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Requirements and reasons for effective inhibition of the anaphase promoting complex activator CDH1.

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Anaphase-promoting complex/cyclosome controls HEC1 stability.

Project description:ObjectiveChromosome segregation during mitosis requires a physically large proteinaceous structure called the kinetochore to generate attachments between chromosomal DNA and spindle microtubules. It is essential for kinetochore components to be carefully regulated to guarantee successful cell division. Depletion, mutation or dysregulation of kinetochore proteins results in mitotic arrest and/or cell death. HEC1 (high expression in cancer) has been reported to be a kinetochore protein, depletion of which, by RNA interference, results in catastrophic mitotic exit.Materials and methods and resultsTo investigate how HEC1 protein is controlled post-translation, we analysed the role of anaphase-promoting complex/cyclosome (APC/C)-Cdh1 in degradation of HEC1 protein. In this study, we show that HEC1 is an unstable protein and can be targeted by endogenous ubiquitin-proteasome system in HEK293T cells. Results of RNA interference and in vivo ubiquitination assay indicated that HEC1 could be ubiquitinated and degraded by APC/C-hCdh1 E3 ligase. The evolutionally conserved D-box at the C-terminus functioned as the degron of HEC1, destruction of which resulted in resistance to degradation mediated by APC/C-Cdh1. Overexpression of non-degradable HEC1 (D-box destroyed) induced accumulation of cyclin B protein in vivo and triggered mitotic arrest.ConclusionAPC/C-Cdh1 controls stability of HEC1, ensuring normal cell cycle progression.

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A novel human Cdh1 mutation impairs anaphase promoting complex/cyclosome activity resulting in microcephaly, psychomotor retardation, and epilepsy.

Project description:The Fizzy-related protein 1 (Fzr1) gene encodes Cdh1 protein, a coactivator of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). Previously, we found that genetic ablation of Fzr1 promotes the death of neural progenitor cells leading to neurogenesis impairment and microcephaly in mouse. To ascertain the possible translation of these findings in humans, we searched for mutations in the Fzr1 gene in 390 whole exomes sequenced in trio in individuals showing neurodevelopmental disorders compatible with a genetic origin. We found a novel missense (p.Asp187Gly) Fzr1 gene mutation (c.560A>G) in a heterozygous state in a 4-year-old boy, born from non-consanguineous Spanish parents, who presents with severe antenatal microcephaly, psychomotor retardation, and refractory epilepsy. Cdh1 protein levels in leucocytes isolated from the patient were significantly lower than those found in his parents. Expression of the Asp187Gly mutant form of Cdh1 in human embryonic kidney 293T cells produced less Cdh1 protein and APC/C activity, resulting in altered cell cycle distribution when compared with cells expressing wild-type Cdh1. Furthermore, ectopic expression of the Asp187Gly mutant form of Cdh1 in cortical progenitor cells in primary culture failed to abolish the enlargement of the replicative phase caused by knockout of endogenous Cdh1. These results indicate that the loss of function of APC/C-Cdh1 caused by Cdh1 Asp187Gly mutation is a new cause of prenatal microcephaly, psychomotor retardation, and severe epilepsy. Read the Editorial Highlight for this article on page 8. Cover Image for this issue: doi: 10.1111/jnc.14524.

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Anaphase-promoting complex/cyclosome-Cdh1 coordinates glycolysis and glutaminolysis with transition to S phase in human T lymphocytes.

Project description:Cell proliferation is accompanied by an increase in the utilization of glucose and glutamine. The proliferative response is dependent on a decrease in the activity of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1 which controls G1-to-S-phase transition by targeting degradation motifs, notably the KEN box. This occurs not only in cell cycle proteins but also in the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), as we have recently demonstrated in cells in culture. We now show that APC/C-Cdh1 controls the proliferative response of human T lymphocytes. Moreover, we have found that glutaminase 1 is a substrate for this ubiquitin ligase and appears at the same time as PFKFB3 in proliferating T lymphocytes. Glutaminase 1 is the first enzyme in glutaminolysis, which converts glutamine to lactate, yielding intermediates for cell proliferation. Thus APC/C-Cdh1 is responsible for the provision not only of glucose but also of glutamine and, as such, accounts for the critical step that links the cell cycle with the metabolic substrates essential for its progression.

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STYK1/NOK affects cell cycle late mitosis and directly interacts with anaphase-promoting complex activator CDH1.

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Coordination of Rho and Rac GTPase function via p190B RhoGAP.

Project description:The Rac GTPase regulates Rho signaling in a broad range of physiological settings and in oncogenic transformation [1-3]. Here, we report a novel mechanism by which crosstalk between Rac and Rho GTPases is achieved. Activated Rac1 binds directly to p190B Rho GTPase-activating protein (RhoGAP), a major modulator of Rho signaling. p190B colocalizes with constitutively active Rac1 in membrane ruffles. Moreover, activated Rac1 is sufficient to recruit p190B into a detergent-insoluble membrane fraction, a process that is accompanied by a decrease in GTP-bound RhoA from membranes. p190B is recruited to the plasma membrane in response to integrin engagement [4]. We demonstrate that collagen type I, a potent inducer of Rac1-dependent cell motility in HeLa cells, counteracts cytoskeletal collapse resulting from overexpression of wild-type p190B, but not that resulting from overexpression of a p190B mutant specifically lacking the Rac1-binding sequence. Furthermore, this p190B mutant exhibits dramatically enhanced RhoGAP activity, consistent with a model whereby binding of Rac1 relieves autoinhibition of p190B RhoGAP function. Collectively, these observations establish that activated Rac1, through direct interaction with p190B, modulates subcellular RhoGAP localization and activity, thereby providing a novel mechanism for Rac control of Rho signaling in a broad range of physiological processes.

| S-EPMC4084715 | biostudies-literature

The anaphase-promoting complex/cyclosome is required for anaphase progression in multinucleated Ashbya gossypii cells.

Project description:Regulated protein degradation is essential for eukaryotic cell cycle progression. The anaphase-promoting complex/cyclosome (APC/C) is responsible for the protein destruction required for the initiation of anaphase and the exit from mitosis, including the degradation of securin and B-type cyclins. We initiated a study of the APC/C in the multinucleated, filamentous ascomycete Ashbya gossypii to understand the mechanisms underlying the asynchronous mitosis observed in these cells. These experiments were motivated by previous work which demonstrated that the mitotic cyclin AgClb1/2p persists through anaphase, suggesting that the APC/C may not be required for the division cycle in A. gossypii. We have now found that the predicted APC/C components AgCdc23p and AgDoc1p and the targeting factors AgCdc20p and AgCdh1p are essential for growth and nuclear division. Mutants lacking any of these factors arrest as germlings with nuclei blocked in mitosis. A likely substrate of the APC/C is the securin homologue AgPds1p, which is present in all nuclei in hyphae except those in anaphase. The destruction box sequence of AgPds1p is required for this timed disappearance. To investigate how the APC/C may function to degrade AgPds1p in only the subset of anaphase nuclei, we localized components and targeting subunits of the APC/C. Remarkably, AgCdc23p, AgDoc1p, and AgCdc16p were found in all nuclei in all cell cycle stages, as were the APC/C targeting factors AgCdc20p and AgCdh1p. These data suggest that the AgAPC/C may be constitutively active across the cell cycle and that proteolysis in these multinucleated cells may be regulated at the level of substrates rather than by the APC/C itself.

| S-EPMC1797942 | biostudies-literature

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